1. Field of the Invention
The present invention relates to a non-radioactive method and a solution or composition for the detection of a ligand and antiligand complex of a DNA or RNA nucleic acid, an antigen, a hapten, a protein, an analyte, an antibody, or an antibody complex wherein the complex is labelled with alkaline phosphatase or a tracer having alkaline phosphatase conjugated thereto, and reacted with bromo-chloro-indolyl phosphate (BCIP), phenazine methosulfate (PMS), and dimethylthiazol diphenyl tetrazolium (MTT) to produce a colored formazan or a color change indicative of the presence of the labelled complex.
2. Description of the Related Art
Labelling a segment of a DNA or RNA nucleic acid, a protein, a hapten, an antigen, an analyte, an antibody or an antibody complex such that the same can be later identified and detected is desirable in many applications, including diagnostic application of probe technologies.
Assay systems which are both rapid and sensitive have been developed to determine the concentration of a substance, for example an analyte, present in low concentration in a fluid sample. Immunoassays depend on the binding of an antigen or hapten to a specific antibody and have been particularly useful because they give high levels of specificity and sensitivity. Such assays may employ a reagent in labelled form referred to as the tracer.
For example, five basic methods of labelling nucleic acids include nick translation, primer extension, methods based on RNA polymerase, end-labelling methods, and direct labelling methods. In many probe technologies, the need for resolution and sensitivity determines the choice of label to DNA or RNA nucleic acid, proteins, or antibodies. Labels for probes are usually radioactive. Biotin is a commonly used non-radioactive label for probes which can be incorporated into polynucleotide enzymatically using biotinylated nucleotide as the substrate. Alternatively, a photoactivatable analogue of biotin upon brief irradiation with visible light may be used to form stable linkages with both single and double stranded nucleic acids. Biotin-labelled probes are detected through a variety of signal generating systems usually using avidin, a glycoprotein with an extremely high affinity for biotin, or streptavidin, an avidin-like protein. Alternatively, it has been known to label DNA with digoxigenin-labelled deoxyuridine triphosphate. After hybridization to the target DNA, the hybrids are detected by enzyme-linked immunoassay using an antibody conjugate such as biotin-conjugated with alkaline phosphatase.
Non-radioactive labels with biotin have lower sensitivity in comparison with radioactive labels. Thus, radioactive probes are used for most commercial applications of hybridization technologies requiring that probes be freshly prepared at regular intervals due to radioisotopes having short half-lives. Radioactive labels also require special safety precautions for the isotopes and proper radioactive waste disposal.
Enzymes have also often been used as labels in immunoassay. In conventional enzyme immunoassay (EIA), an enzyme is covalently conjugated with one component of a specifically binding antigen-antibody pair, and the resulting enzyme conjugate is reacted with a substrate to produce a signal which is detected and measured. The signal may be a colour change, detected with the naked eye or by a spectrophotometric technique, or may be conversion of the substrate to a product detected by fluorescence.
A convenient format for EIA is solid phase immunoassay in which one of the assay reagents is immobilized on a solid support usually in the form of a dip stick, the inside wall of a test tube or cuvette, the well of a microtiter plate, or a microporous membrane. The final step in most membrane EIA procedures is contacting a color developing reagent, such as a chromogen, with the membrane. The chromogen reacts with enzyme captured on the membrane to produce a colored product which may be detected as evidence of the presence of analyte or measured as evidence of the concentration of analyte.
Tetrazolium salts have been used for analytical purposes in the detection of reduced nicotinamideadenine dinucleotide (NADH) wherein the transference of hydrogen is catalyzed not only by enzymes, such as diaphorase, but also by 5-methylphenazinium methylsulphate (PMS) or similar substances, to thereby form deep colored formazans as a reduction indicator. Therefore, appropriate processes have been developed in this way to detect a series of substances which are important in analytical chemistry, via the NADH produced as an intermediate. Tetrazolium salts conventionally employed in dehydrogenase procedures include 3-(4.5'-dimethylthiazolyl-2)-2,4-diphenyltetrazolium bromide (MTT), 2-(p-iodophenyl)-3-(p-nitrophenyl)-5-phenyl-tetrazolium chloride (INT), 2,2',5,5'-tetra-(p-nitrophenyl)-3,3-(3-dimethoxy-4-diphenylene)-ditetrazol ium chloride (TNBT), 2,2'-di-(p-nitrophenyl)-5.5'-diphenyl-3,3'-dimethoxy-4,4'-diphenylene)-dit etrazolium chloride (NBT), 2,2'-p-diphenylene-3,3',5.5'-tetraphenylditetrazolium chloride (neotetrazolium chloride) (NT) and 2,3,5-triphenyltetrazolium chloride (TT).
U.S. Pat. Nos. 4,613,569 and 4,867,196 to Giesler et al. are directed to a stabilized composition of tetrazolium salts containing one to ten moles of a complex-forming acid, such as boric acid or organic hydroxypolylcarboxylic acid, which is soluble in polar solvents per mole of tetrazolium salt. The stabilizing agents are employed in previously known test systems in which the tetrazolium salts are used as indicators such as dehydrogenase procedures involving the detection of lactic acid with lactate dehydrogenase, alcohol with alcohol dehydrogenase, glycerol with glycerol dehydrogenase, glucose with glucose dehydrogenase, acetaldehyde with acetaldehyde dehydrogenase, as well as further systems which can be coupled to the above system.
The present invention provides for a non-radioactive method of detecting (as well as a solution and composition therefor) a ligand and antiligand complex labelled with alkaline phosphatase or a tracer having alkaline phosphatase conjugated thereto which comprises reacting the complex with bromo-chloro-indolyl phosphate (BCIP), phenazine methosulfate (PMS) and dimethylthiazol diphenyl tetrazolium (MTT) and allowing the reaction to proceed to reduce the dimethylthiazol diphenyl tetrazolium (MTT) and form a colored formazan or produce a color change indicative of the presence of the labelled complex.